鸭疫里默氏杆菌红霉素耐药机制的研究

发布时间：2018-04-27 04:27

  本文选题：鸭疫里默氏杆菌 + 红霉素抗性　； 参考：《山东农业大学》2015年硕士论文

【摘要】：鸭疫里默氏杆菌是黄杆菌科鸭疫里默氏杆菌属的代表种,黄杆菌科是拟杆菌门最大的一个分支,目前鸭疫里默氏杆菌已成为危害养鸭业的主要病原之一,不仅感染鸭、火鸡,其他畜禽如鸡、鹅等也容易受到感染。病鸭死亡率高、生长迟缓、饲料转化效率低、品质下降、疾病治疗等,给养殖业造成了巨大的经济损失。控制该病的方法主要是注射疫苗和应用抗菌药物,其中红霉素已在临床上成功应用于该病,但是近些年红霉素治疗失败的案例已经被多次报道。近年来分离出来的具有红霉素抗性的鸭疫里默氏杆菌比例逐渐增加,而鸭疫里默氏杆菌的红霉素抗性机制还未被报道。故本试验重点研究鸭疫里默氏杆菌对红霉素的耐药机制。本研究以先采用微量肉汤稀释法分别检测了1996年至2014年本实验室自临床病死鸭分离的79株鸭疫里默氏杆菌及一株鸭疫里默氏杆菌型菌株ATCC11845的最小抑菌浓度(MIC),结果表明检测的80株鸭疫里默氏杆菌中有43株具有红霉素抗性,占53.8%,37株为红霉素敏感菌株,占46.2%。此外,2005—2014年自病鸭分离的56株鸭疫里默氏杆菌中有41株为红霉素耐药菌,占73.2%(41/56);而1996—2004年自病鸭分离的24株鸭疫里默氏杆菌中只有2株为红霉素耐药菌。为了确定所携带红霉素基因的类型,通过PCR检测耐药菌株的ermF基因,结果表明,具有红霉素抗性的43株鸭疫里默氏杆菌中有30株携带有ermF或ermFU基因,MIC在32~2048mg/L;具有emF+红霉素抗性的30株菌中有27株携带ermFU基因,占90%(27/30);另外3株菌携带有ermF基因。另外,其余13株红霉素抗性的鸭疫里默氏杆菌携带有ereD基因,MIC在4~16 mg/L。克隆不同ermF+阳性菌株的ermF/ermFU基因并进行序列分析,结果表明其氨基酸同源性高达98.5%-100%。对鸭疫里默氏杆菌菌株HXb2、GuiZ-1、YXb15、HGb1、NJ-4、JY-6、YZ-1、ZJb2分别通过基因组步移法扩增ermF基因及其侧翼序列,结果显示,鸭疫里默氏杆菌与CTnDOT的ermFU基因的上游序列有85.8%-88.7%的同源性,因此推测ermFU基因可能是来自CTnDOT转座子元件;而鸭疫里默氏杆菌的ermF基因的上游(-1至-600)与转座子Tn4351中的ermF基因相应区域相比较同源性较高,仅有几个核苷酸的差异,因此推测ermF基因可能来自于转座子Tn4351。此外,序列分析显示ermF、ermFU和ereD基因存在于鸭疫里默氏杆菌基因组的多重耐药区(MRR)。为了进一步确定ermF、ermFU和ereD基因能否引起鸭疫里默氏杆菌的红霉素耐药,将分别携带有ermF、ermFU和ereD基因盒的重组穿梭质粒转入到红霉素敏感菌株CH3中。结果显示,三株重组菌CH3(pRES-HXb2-ermFU),CH3(pRES-YZ-1-erm F)和CH3(pRES-SX-ere D)均具有红霉素抗性,其中携带有ermF或ermFU的两个重组菌株CH3(pRES-HXb2-ermFU)和CH3(pRES-YZ-1-ermF)对林可霉素具有较高的抗性,而CH3(pRES-SX-ereD)对林可霉素同野生株一样对红霉素敏感。荧光定量PCR显示,三株重组菌CH3(pRES-HXb2-ermFU),CH3(pRES-YZ-1-erm F)和CH3(pRES-SX-ere D)中ermFU、ermF和ereD的m RNA相对表达量分别较野生株HXb2、YZ-1和SX高3.3、80和6.7倍。以上结果表明,鸭疫里默氏杆菌多重耐药区(DRR)中的ermF、ermFU或ereD基因可引起鸭疫里默氏杆菌的红霉素耐药。
[Abstract]:The Bacillus pestis are the representative species of the family of the family of the bacilliaceae, and the family of the family is the largest branch of the bacteriobacteriaceae. At present, the bacilli has become one of the main pathogens that harm the duck industry. It is not only infected with ducks, turkeys, but also other livestock and poultry such as chicken and goose. The low efficiency of feed conversion, the decline of the quality and the treatment of disease have caused great economic loss to the breeding industry. The main methods to control the disease are the injection of vaccines and the application of antibiotics. In recent years, erythromycin has been successfully applied to the disease, but in recent years the cases of erythromycin treatment have been reported many times. The ratio of erythromycin resistance to erythromycin gradually increased and the mechanism of erythromycin resistance was not reported. This study focused on the mechanism of drug resistance to erythromycin. This study was conducted from 1996 to 2014 by micro broth dilution method. The minimum inhibitory concentration (MIC) of 79 strains of M. ducks isolated from dead ducks and one strain of the strain of the strain ATCC11845 showed that 43 of the 80 strains had erythromycin resistance, 53.8%, 37 erythromycin sensitive, 56 from 2005 to 2014, and 56 strains isolated from the disease duck from 2005 to 2014. 41 strains were erythromycin resistant bacteria, accounting for 73.2% (41/56), while only 2 strains of erythromycin resistant strains isolated from 1996 to 2004 were erythromycin resistant strains. In order to determine the type of erythromycin gene carried, the ermF basis of the resistant strain was detected by PCR. The results showed that 43 strains of erythromycin resistance were found in the duck epidemic. 30 of meribacilli were carried with ermF or ermFU genes and MIC in 32~2048mg/L; 27 of the 30 strains with emF+ erythromycin resistance were carrying ermFU genes, accounting for 90% (27/30), and the other 3 strains carried ermF gene. In addition, the remaining 13 strains of erythromycin resistance were carried with ereD gene, MIC in 4~16 mg/L.. The ermF/ermFU gene of the strain and sequence analysis showed that its amino acid homology was up to 98.5%-100%. for the strain HXb2, GuiZ-1, YXb15, HGb1, NJ-4, JY-6, YZ-1, and ZJb2 respectively. The ermF gene and its flanking sequence were amplified by the genome step method respectively. The sequence has the homology of 85.8%-88.7%, so it is presumed that the ermFU gene may come from the CTnDOT transposon element, while the upstream of the ermF gene of riemulibacillus duck pestis (-1 to -600) is more homologous to the corresponding region of the ermF gene in the transposon Tn4351, with only a few nucleotide differences. Therefore, it is presumed that the ermF gene may come from the transposing. In addition, sequence analysis showed that ermF, ermFU and ereD genes existed in the multidrug-resistant region of the genome of reeminorus pestis (MRR). In order to further determine whether the ermF, ermFU and ereD genes could cause erythromycin resistance to reeminorus pestis, the recombinant shuttle plasmid containing ermF, ermFU and ereD gene boxes would be transferred to erythromycin, respectively. The results showed that three recombinant strains CH3 (pRES-HXb2-ermFU), CH3 (pRES-YZ-1-erm F) and CH3 (pRES-SX-ere D) all had erythromycin resistance, and the two recombinant strains carrying ermF or ermFU were resistant to lincomycin. The fluorescent quantitative PCR showed that the relative expression of three recombinant strains CH3 (pRES-HXb2-ermFU), CH3 (pRES-YZ-1-erm F) and CH3 (pRES-SX-ere D) was higher than that of wild plants, respectively, and 6.7 times higher than that of wild strains. F, ermFU or ereD genes can cause erythromycin resistance in R.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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